This study investigated the effects of a 14-day spaceflight on bone mass, density and microarchitecture in weight bearing (femur and humerus) and non-weight bearing (2(nd) lumbar vertebra and calvarium) bones in the context of ovarian hormone insufficiency. 12-week-old Fisher 344 rats were ovariectomized 2 weeks before flight and randomized into one of three groups: 1) baseline (n = 6), 2) ground control (n = 12) or 3) spaceflight (n = 12). Additional ground-based ovary-intact rats provided age-matched reference values at baseline (n = 8) and landing (n = 10). Ovariectomy resulted in bone- and bone compartment-specific deficits in cancellous bone volume fraction. Spaceflight resulted in lower cortical bone accrual in the femur but had no effect on cortical bone in the humerus or calvarium. Cancellous bone volume fraction was lower in flight animals compared to ground control animals in lumbar vertebra and distal femur metaphysis and epiphysis; significant differences were not detected in the distal humerus. Bone loss (compared to baseline controls) in the femur metaphysis was associated with lower trabecular number, whereas trabecular thickness and number were lower in the epiphysis. In summary, the effect of spaceflight on bone microarchitecture in ovariectomized rats was bone-and bone compartment-specific but not strictly related to weight bearing.